Using a combined ATACseq and RNAseq approach our studies have provided the first evidence that changes in chromatin accessibility are crucial to the differentiation state-specific expression of a wide variety genes essential to the transition from lens epithelial to fiber cells. These studies also identified key DNA regulatory regions and transcription factor binding sites likely to regulate a wide-range of lens genes, most importantly FOXO4. Genes with requisite roles in lens cell differentiation that contain a consensus DNA binding sequence for FOXO4 include EPHA2, NrCAM, ?-crystallin, Notch1 and FYCO1. The FOXO family, including FOXO4, is regulated by the PI3K/Akt signaling pathway. Phosphorylation of FOXO4 by PI3K/Akt sequesters it in the cytoplasm and suppression of PI3K/Akt signaling is required for FOXO4 import to the nucleus for its role in regulating gene expression. The function of FOXO4 has never been examined in the lens. We propose to establish the role of PI3K/AKT regulation of FOXO4-dependent gene expression in lens fiber cell differentiation. This will be accomplished by 1) identifying the requirement for PI3K/Akt inhibition for the nuclear translocation of FOXO4 in the transition of lens epithelial to fiber cells; 2) establishing the link between PI3K/Akt inhibition and the expression of lens differentiation-specific genes containing FOXO4 binding sequences; 3) demonstrating the binding of FOXO4 to distinct chromatin accessible DNA regulatory regions in genes crucial to lens differentiation using targeted-CHIP assays; and 4) establishing the spectrum, range and spatial expression patterns of genes regulated by FOXO4 during lens cell differentiation. We also discovered that the PI3K/Akt signaling axis plays an essential role in regulating the timing and mechanism that removes mitochondria, ER and Golgi from the central light path and that multiple PI3K-downstream signaling pathways are required for the process of eliminating nuclei to form the lens Organelle Free Zone (OFZ). This process is required to create a mature lens capable of focusing light images on the retina. We will explore how PI3K signaling pathways regulate the elimination of nuclei and other organelles to form the lens Organelle Free Zone in studies aimed at 1) identifying the functions of individual PI3K p110 catalytic subunits in regulating formation of the OFZ; 2) confirming that the induction of autophagy following inhibition of the PI3K/Akt signaling axis is responsible for the removal of mitochondria, ER and Golgi from the developing lens; 3) determining the link between inactivation of different PI3K signaling pathways and activation of the mechanistic targets required to eliminate nuclei to form the OFZ; and 4) investigating the potential link between the ring of Akt activity at the border of cortical and nuclear fiber cells and the regulation of the outer boundary of the OFZ.